Cloning, characterization, and molecular application of a beta-agarase gene from Vibrio sp strain V134

V134, a marine isolate of the Vibrio genus, was found to produce a new beta-agarase of the GH16 family. The relevant agarase gene agaV was cloned from V134 and conditionally expressed in Escherichia coli. Enzyme activity analysis revealed that the optimum temperature and pH for the purified recombinant agarase were around 40 degrees C and 7.0. AgaV was demonstrated to be useful in two aspects: first, as an agarolytic enzyme, the purified recombinant AgaV could be employed in the recovery of DNA from agarose gels; second, as a secretion protein, AgaV was explored at the genetic level and used as a reporter in the construction of a secretion signal trap which proved to be a simple and efficient molecular tool for the selection of genes encoding secretion proteins from both gram-positive and gram-negative bacteria.

Natural bromophenols from the marine red alga Polysiphonia urceolata (Rhodomelaceae): Structural elucidation and DPPH radical-scavenging activity

Three new natural occurring bromophenols, 3-(3-bromo-4,5-dihydroxyphenyl)-2-(3,5-dibromo-4-hydroxyphenyl)propionic acid (1), (E)-4-(3-bromo-4,5-dihydroxyphenyl)-but-3-en-2-one (2), and (3,5-dibromo-4-hydroxyphenyl) acetic acid butyl ester (3), together with one known bromophenol, 1,2-bis(3-bromo-4,5-dihydroxyphenyl)ethane (4), were isolated and identified from the marine red alga Polysiphonia urceolata. The structures of these compounds were elucidated by extensive analysis of ID and 2D NMR and IR spectra and MS data. Each of the isolated compounds was evaluated for scavenging alpha,alpha-diphenyl-beta-picrylhydrazyl (DPPH) radical activity and all of them exhibited significant activity with IC50 values ranging from 9.67 to 21.90 mu M, compared to the positive control, a well-known antioxidant butylated hydroxytoluene (BHT), with IC50 83.84 mu M. (C) 2007 Elsevier Ltd. All rights reserved.

Molecular cloning and functional analysis of cathepsin B in nutrient metabolism during larval development in Meretrix meretrix

Seed rearing is an important part in large scale clam culture industry. Since the nutritional history affects early development in bivalve, the condition of larval nutrition plays a key role in successful seed rearing. So far, the molecular mechanism of nutrient uptake in bivalve larvae is unclear. As one of the important proteolytic enzymes, cathepsin B of several organisms has been reported to be involved in digestion. We intended to analyze whether cathepsin B is involved in larval nutrient metabolism in the economic bivalve, clam Meretrix meretrix. The full length of M. meretrix cathepsin B (MmeCB) cDNA was cloned, which is 1647 bp with an open reading frame of 1014 bp. The deduced amino acid sequence encoded a preproenzyme of 337 residues with Cys-114, His-282 and Asn-302 composing cathepsin B activity center. The temporal and spatial expressions of MmeCB mRNA were examined from trochophore to post larva stages by whole mount in situ hybridization. In trochophore stage, no detectable signal was found. In the later three stages, MmeCB mRNA was detected in the digestive gland, suggesting a possible role of MmeCB in digestion. Moreover, MmeCB mRNA was also observed in the epidermal cells in D-veligers. Cathepsin B specific inhibitor (CA074 methyl ester) was applied to block the activity of cathepsin B in unfed larvae. The average shell lengths of treated larvae were smaller than that in control groups. The results of mRNA epidermal distribution and inhibitor treatment in D-veligers indicated that MmeCB may be also associated with other pathway of nutrient metabolism in larval epidermis. The overall results in this paper revealed that MmeCB might play a role in larval nutrient metabolism. (C) 2008 Elsevier B.V. All rights reserved.

Extracellular hydrolytic enzyme screening of culturable heterotrophic bacteria from deep-sea sediments of the Southern Okinawa Trough

The Southern Okinawa Trough is an area of focused sedimentation due to particulate matter export from the shelf of the East China Sea and the island of Taiwan. In order to understand the geomicrobiological characteristics of this unique sedimentary environment, bacterial cultivations were carried out for an 8.61 m CASQ core sediment sample. A total of 98 heterotrophic bacterial isolates were characterized based on 16S rRNA gene phylogenetic analysis. These isolates can be grouped into four bacterial divisions, including 13 genera and more than 20 species. Bacteria of the gamma-Proteobacteria lineage, especially those from the Halomonas ( 27 isolates) and Psychrobacter ( 20 isolates) groups, dominate in the culturable bacteria assemblage. They also have the broadest distribution along the depth of the sediment. More than 72.4% of the isolates showed extracellular hydrolytic enzyme activities, such as amylases, proteases, lipases and Dnases, and nearly 59.2% were cold-adapted exoenzyme-producers. Several Halomonas strains show almost all the tested hydrolases activities. The wide distribution of exoenzyme activities in the isolates may indicate their important ecological role of element biogeochemical cycling in the studied deep-sea sedimentary environment.

羟基自由基降解琼胶及其产物的活性研究

琼胶是一种从石花菜等红藻中提取的，目前生产工艺和结构等方面研究比较成熟的海藻多糖，广泛应用于医药、仪器等行业。但是，海藻多糖因为具有分子量大，粘度大，溶解度较小的等特点，而使其应用范围受到限制。利用降解的手段对其进行修饰，降低分子量和粘度，改善溶解性，可以拓展其应用范围。并且根据文献报道，琼 胶寡糖具有一些特殊的生物活性，如抗氧化性，抗炎症等。因此，对琼胶降解的研究具有生要意义。本研究中，为了选择一种合适的降解方法，进行了几种水解方法的尝试，其中包括在不同湿度和酸度下盐酸水解，过氧化氢和醋酸催化水解，Fenton体系羟基自由基降解。对于酸水解和Fenton体系氧化还原降解方法，通过粘度法对反应的速度进行了比较，表明氧化还原降解反应中琼胶的粘度降低比较快，并且具有代表性和新意，确定为本实验的降解琼胶的方法并对氧化还原降解所得的产物进行了活性实验。通过模仿自然界普遍存在的氧化还原降解反应，利用Vc诱导的Fenton体系产生的羟基自由基氧化还原降解琼胶得到低分子量的琼胶。降解产物经过高速离心、60％乙醇沉淀，除去分子量比较大的降解产物和磷酸盐，得到可溶于60％乙醇的分子量估计小于3000的降争产物，其产率为85％。利用经Sephadex-G25凝胶色谱分离所香的不同分子量的级分进行分子量和α－葡萄糖苷酶抑制活性关系的实验。降解产物对α－葡萄糖苷酶的抑制率和各级分的浓度呈线性正相关，并且各级分的IC_(50)则随着分子量的降低而降低。另外，对所得的降解产物混合物进行了红外吸收光谱、质子去偶核磁共震碳谱和负离子基质辅助激光诱导－飞行时间质谱结构分析。结果表明，氧化还原降解反应的专一性差，在得到寡糖的同时，在光谱图中出现一些比较复杂的副产物的结构信息。最后，根据MTT法的原理，以有体皮肤成纤维细胞为材料，通过紫外线辐射产生自由基造成氧化损伤，研究降解产物对成纤维细胞的保护作用。当无紫外线辐射时，降解产物对成纤维细胞具有显著的促进生长增殖作用：当经UVa、UBb辐射时则可以显著地表现出对损伤的保护作用，并且这种促进生长和保护作用呈显著的量效关系，表明降解产物具有清除基自由基的作用。但是，因为氧化还原降解以应的机理尚不十分明的以及琼羟胶的特殊结构，使得反应的副产物很难预测，也就使得分离工作难以进行，所以，根据目前所得的信息，尚不能确定是降解产物的什么级分产生的以上两种生物活性。

三种海洋红藻和两株放线菌次级代谢产物研究

海洋是一个巨大的天然产物宝库，约占地球表面积70%的海洋蕴藏着80%的生物资源。由于海洋生态环境的特殊性，导致海洋生物能够产生大量结构独特多变和活性特殊多样的代谢产物。我国海域辽阔，海洋资源丰富，为寻找结构新颖、生理活性独特的先导化合物，加强对海洋资源的开发利用，本论文对中国沿海的三种海洋红藻和两株放线菌次生代谢产物以及生物活性进行研究，为新药研究与开发提供模式结构和药物前体。 对红藻似瘤凹顶藻Laurencia similis乙酸乙酯萃取物进行分离纯化，从中得到单体化合物35个，通过波谱学方法（IR、MS、NMR等）鉴定了他们的结构。分别为：2, 2, 5, 5, 6, 6-sixibromo-3, 3-bi-1H-indole (1)，3,5-dibromo- 1-methyl-indole (2)，3',5',6,6'-tetrabromo-2,4-dimmethyldiphenyl ether (3)，1,2,5- tribromo-3-bromoamino-7-bromomethylnaphthalene (4)，2,5,8-tribromo-3-bromo- amino-7-bromomethylnaphthalene (5)，2,5,6-tribromo-3-bromoamino-7-bromo- methylnaphthalene (6), 2,5,6,5',6'-pentabromo-3,4,3',4'-tetramethoxybenzophenone (7), (4E)-1-bromo-5-[(1'S*,3'R*)-3'-bromo-2',2'-dimethyl-6'-methylenecyclohexyl] -3-methylpent-4-ene-2,3-diol (8)，4-hydroxy-Palisadin C (9)，Isopalisol (10)，Luzonensol (11)，Palisadin B (12)，Aplysistatin (13)，Palisadin A (14)，5-Acetoxypalisadin B (15)，Aristolan-1(10)- en-9-ol (16)，Aristol-8-en-1-one (17)，Aristolan-9-en-1-one (18)，Aristolan-1(10)-en- 9-one (19)，Aristofone (20)，Aristolan-1(10)-8-diene (21)，Aristolan-1,9-diene (22)，10-Hydroxyaristolan-9-one (23)，7,11,15-trimethyl-3-methylene-hexadecan-1,2-diol (24)，3β-Hydroxyergosta- 5,24(28)-dien-7-one (25)，Isofucosterol (26)，β-sitosterol (27)，豆甾-4-烯-3α,6β-二醇 (28)，Cholesta-5-en-3β-ol (29)，Stigmasterol (30)，2,3,5,6-四溴-吲哚 (31)，2,3,6-tribromo-1H-indole (32)，3,5,6-tribromo-1-methylindole (33)，3,5,6-tribromo -1H-indole (34)，2,3,5-tribromo-1-methylindole (35)，其中化合物1-9为新化合物，化合物10-15、20和化合物24-30均为首次从该种海藻中得到。对新化合物1-9进行PTP1B酶抑制剂活性筛选，新化合物1、3、7显示强的PTP1B酶抑制活性。 对红藻齐藤凹顶藻Laurencia saitoi乙酸乙酯萃取物进行分离纯化，从中得到单体化合物11个，通过波谱学方法（IR、MS、NMR等）鉴定了他们的结构，分别为：2-hydroxyl-Luzofuranone (1)，2-hydroxyl-Luzofuranone B (2)，4-hydroxyl-Palisudin C (3)，2-bromo-γ-ionone (4)，Aplysistatin (5)，5-Acetoxypalisadin B (6)，Palisadin B (7)，Palisadin A (8)，Pacifigorgiol (9)，豆甾-4-烯-3α,6β-二醇 (10)，2, 3, 5, 6-四溴-吲哚 (11)，其中化合物1-4为新化合物，所有化合物均为首次从该种海藻中得到。通过MTT法对分离得到的新化合物1-4进行肿瘤细胞毒活性筛选，结果显示4个新化合物对所测肿瘤细胞株均无明显的活性。 对红藻瘤状软骨凹顶藻Chondrophycus papillous乙酸乙酯萃取物进行分离纯化，从中得到单体化合物5个，通过波谱学方法（MS、NMR等）鉴定了他们的结构，分别为邻苯二甲酸二丁酯 (1)，邻苯二甲酸二异辛酯 (2)，胆甾醇 (3)，3,7,11,15-tetramethyl-hexadec-2-en-1-ol (4)，4-羟基苯甲醛 (5)，所有化合物均为首次从该种海藻中得到。 对海洋放线菌M159乙酸乙酯萃取物进行分离纯化，从中得到单体化合物13个，通过波谱学方法（MS、NMR等）鉴定了他们的结构，分别为：5-(4',6'-dihydroxy-6-methyloctyl)furan-2(5H)-one (A)，phenethyl alcohol (1)，4-羟基苯甲醛(2)，anthranilic acid (3)，4-Hydroxy-3-methoxy- phenyl-propionic acid (4)，5-(6,7-dihydroxy-6-methyloctyl)furan-2(5H)-one (5)，p-Hydroxyphenylethyl alcohol (6)，3-Indoleacrylic acid (7)，Indol-3-carboxylic acid (8)，Adenine cordyceposide (9)，腺嘌呤核苷(10)，尿嘧啶核苷(11)，Thymidine (12)，其中化合物A为新化合物。所有化合物均为首次从该株放线菌中得到。 对海洋放线菌L211乙酸乙酯萃取物进行分离纯化，从中得到单体化合物15个，通过波谱学方法（MS、NMR等）鉴定了7个结构，分别为：spatozoate (1)，anthranilic acid (2)，3-Indolylethanol (3)，1-Acetyl-β-carbolin (4)，p-Hydroxyphen- ylethyl alcohol (5)，Indole-3-acetic acid (6)，Indol-3-carboxylic acid (7)，所有化合物均为首次从该株放线菌中得到。

Bromophenols from the red alga Rhodomela confervoides

Six new bromophenols, 3-bromo-4,5-bis(2,3-dibromo-4,5-dihydroxybenzyl)pyrocatechol (1), 2,2',3-tribromo-3',4,4',5-tetrahydroxy-6'-hydroxymethyldiphenylmethane (2), 2,2',3-tribromo-3',4,4',5-tetrahydroxy-6'-ethyloxymethyldiphenylmethane (3),(+/-)-2-methyl-3-(2,3-dibromo-4,5-dihydroxyphenyl)propylaldehyde (4), (+/-)-2-methyl-3-(2,3-dibromo-4,5-dihydroxyphenyl)propylaldehyde dimethyl acetal (5), and 3-bromo-4,5-dihydroxybenzoic acid methyl ester (6), together with eight known bromophenols, 3-bromo-4,5-dihydroxybenzaldehyde (7), 2,3-dibromo-4,5-dihydroxybenzyl alcohol (lanosol, 8), 2,3-dibromo-4,5-dihydroxybenzyl methyl ether (9), 2,3-dibromo-4,5-dihydroxybenzyl ethyl ether (10), 2,3-dibromo-4,5-dihydroxybenzylaldehyde (11), bis(2,3-dibromo-4,5-dihydroxybenzyl) ether (12), 3-bromo-4-(2,3-dibromo-4,5-dihydroxybenzyl)-5-methoxymethylpyrocatechol (13), and 2,2',3,3'-tetrabromo-4,4',5,5'-tetrahydroxydiphenyl methane (14), were isolated from the red alga Rhodomela confervoides. Their structures were elucidated by chemical and spectroscopic methods including IR, HRFABMS, and 1D and 2D NMR techniques.

Bromophenols coupled with nucleoside bases and brominated tetrahydroisoquinolines from the red alga Rhodomela confervoides

Three new bromophenols C-N coupled with nucleoside base derivatives (1-3) and three new brominated 1,2,3,4-tetrahydroisoquinolines (5-7, together with a new brominated tyrosine derivative (4, have been isolated from polar fractions of an ethanolic extract of the red alga Rhodomela confervoides. By spectroscopic and chemical methods including HRMS and 2D NMR data, their structures were determined as 7-[3-bromo-2-(2,3-dibromo-4,5-dihydroxybenzyl)-4,5-dihydroxybenzyl]-3,7-dihydro-1H-purine-2,6-dione (1), 7-(2,3-dibromo-4,5-dihydroxybenzyl)-3,7-dihydro-1H-purine-2,6-dione (2, 9-[3-bromo-2-(2,3-dibromo-4,5-dihydroxybenzyl)-4,5-dihydroxybenzyl]adenine (3), (-)-8S-(3-bromo-5-hydroxy-4-methoxy)phenylalanine (4), (-)-3S-8-bromo-6-hydroxy-7-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (5), methyl (-)-3S-8-bromo-6-hydroxy-7-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (6), and methyl (-)-3S-6-bromo-8-hydroxy-7-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (7). Compounds 5-7 were semisynthesized by using 4 as the starting material.

Bromophenol derivatives from the red alga Rhodomela confervoides

Eight new bromophenol derivatives, 2,3-dibromo-4,5-dihydroxybenzyl methyl sulfoxide (1), 4-(2,3-dibromo-4,5-dihydroxyphenyl)-3-butene-2-one (2), 2-(3-bromo-5-hydroxy-4-methoxyphenyl)-3-(2,3-dibromo-4,5-dihydroxyphenyl)propionic acid (3), 2-(3-bromo-5-hydroxy-4-methoxyphenyl)-3-(2,3-dibromo-4,5-dihydroxyphenyl)propionic acid methyl ester (4), 2-phenyl-3-(2,3-dibromo-4,5-dihydroxyphenyl)propionic acid (5), 4'-methoxy-2",3',3"-tribromo-4",5',5"-trihydroxydiphenylacetic acid (6), and 3-bromo-5-hydroxy-4-methoxyphenylacetic acid (7) and its methyl ester (8), together with a known bromophenol, 3-bromo-5-hydroxy4-methoxybenzoic acid (9), were isolated from the red alga Rhodomela confervoides. Their structures were elucidated by spectroscopic methods including IR, EIMS, FABMS, ESIMS, HRFABMS, HRESIMS, 1D and 2D NMR, and single-crystal X-ray structure analysis. Compounds 1-4, 8, and 9 were found inactive against several human cancer cell lines and microorganisms.

A new brominated phenylpropylaldehyde and its dimethyl acetal from red alga Rhodomela confervoides

A new brominated phenylpropylaldehyde and its dimethyl acetal together with a new natural brominated phenol were isolated from Rhodomela confervoides. Their structrues were elucidated as 2-methyl-3-(2,3-dibromo-4,5-dihydroxyphenyl)propylaldehyde, 2-methyl-3-(2,3-dibromo-4,5-dihydroxyphenyl) propylaldehyde dimethyl acetal and 3-bromo-4,5-dihydroxybenzoic acid methyl ester by spectroscopic techniques including IR, HRFABMS, ID and 2DNMR experiments.

Adsorption and Corrosion Inhibition Behavior of Mild Steel by One Derivative of Benzoic-Triazole in Acidic Solution

A newly synthesized benzoic-triazole derivative 3,5-dimethylbenzoic acid [1,2,4]triazol-l-ylmethyl ester (DBT) was investigated as a corrosion inhibitor of mild steel in 1 M HCl solution using weight loss measurements, potentiodynamic polarization, SEM, and EIS methods. The results revealed that DBT was an excellent inhibitor, and the inhibition efficiencies obtained from weight loss and electrochemical experiments were in good agreement. Using the potentiodynamic polarization technique, the inhibitor was proved to have a mixed-type character for mild steel by suppressing both anodic and cathodic reactions on the metal surface. The number of water molecules (X) replaced by a molecule of organic adsorbate was determined from the Flory-Huggins, Dhar-Flory-Huggins, and Bockris-Swinkels substitutional adsorption isotherms applied to the data obtained from the gravimetric experiments performed on a mild steel specimen in 1 M HCl solution at 298 K.

Electrochemical, quantum chemical and SEM investigation of the inhibiting effect and mechanism of ciprofloxacin, norfloxacin and ofloxacin on the corrosion for mild steel in hydrochloric acid

The inhibiting effect and mechanism of 1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline carboxylicacid(ciprofloxacin), 1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline carboxylic acid (norfloxacin) and (-)-(S)-9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-7 H-pyrido(1,2,3-de)-1,4-benzoxazine-6 carboxylic acid (ofloxacin) on the corrosion of mild steel in 1 mol/L HCl have been studied using electrochemical method, quantum chemical method and SEM at 303 K. The potentiodynamic results showed that these compounds suppressed both cathodic and anodic processes of mild steel corrosion in 1 mol/L HCl. The impedance spectroscopy showed that R-p values increased, and C-dl values decreased with the rising of the working concentration. Quantum chemical calculation showed that there was a positive correlation between some inhibitors structure properties and the inhibitory efficiency. The inhibitors function through adsorption followed Langmuir isotherm, and chemisorption made more contribution to the adsorption of the inhibitors on the steel surface compared with physical adsorption. SEM analysis suggested that the metal had been protected from aggressive corrosion because of the addition of the inhibitors.

Chemical Constituents of Nitraira tangutorum Seed from Qaidam Basin

Six compounds were isolated from the 75% ethanol extract of Nitraria tangutorum seed.On the basis of spectroscopic methods including 1H NMR,13C NMR and ESI-MS and comparison with literature,their structures were elucidated as daucosterol(1),4-hydroxypipecolic acid(2),quercetin(3),allantoin(4),1,2,3,4-tetrahydro-1-methyl-β-carboline-3-carboxylic acid(5) and L-tyrosine(6).Compounds 1,2,3,5 and 6 were isolated from Nitraria tangtorum for the first time.

毛细管电泳法分析唐古特白刺种子中两种生物碱

利用毛细管电泳法分离测定唐古特白刺种子中的尿囊素和吲哚生物碱1-methyl-1，2，3，4-tetrahydro-β-earboline-3-carboxylic acid（MTCCA），所用毛细管规格为48，5cm×50μm i．d．，DAD检测波长220nm，最佳分离条件：电压19kV，分离温度25℃，背景电解质为含有32mmol／LSDS，体积分数10．0％乙腈的32mmol／L硼酸溶液，pH10．0。MTCCA与尿囊素分别在350．0～11.0μg／mL和112．5～3．5μg／mL质量浓度范围内与电泳峰面积呈现良好线性关系，检出限分别为5．0μg/mL和2.5μg／mL。对标准品进行6次测定，迁移时间的RSD为1.1％和1．4％，峰面积的RSD为2.3％和0.82％。

Chemical constituents from euphorbia wallichii

Eleven known compounds were isolated from the roots of Euphorbia wallichii for the first time. They were elucidated to be three triterpenoids, β-amyrin (1), β-amyrin acetate (2) and 3β-acetoxy-lupenol (3), one nor-triterpene peroxide baccatin (4), two caffeic esters (5a, 5b), palmitic acid-2,3-dihydroxypropanenyl ester (6), palmitic acid (7), scopoletin (8), β-sitosterol (9) and daucosterol (10) on the basis of spectral methods. Among them, compound 5a, 5b were reported firstly in the spurge family. And the NMR assignments of compounds 5a and 5b were given for the first time.